NFAM1, an immunoreceptor tyrosine-based activation motif-bearing molecule that regulates B cell development and signaling

Abstract

A functional cDNA cloning system was developed by using a retrovirus library encoding CD8-chimeric proteins and a nuclear factor of activated T cells (NFAT)-GFP reporter cell line to identify molecules inducing NFAT activation. By using this strategy, NFAT activating molecule 1 (NFAM1) was cloned as an immunoreceptor tyrosine-based activation motif (ITAM)-bearing cell surface molecule belonging to the Ig superfamily and is predominantly expressed in spleen B and T cells. NFAM1 crosslinking induced ITAM phosphorylation, ZAP-70/Syk recruitment, NFAT activation, and cytokine production. In vivo overexpression of NFAM1 in bone marrow chimeras and transgenic mice induced severe impairment of early B cell development in an ITAM-dependent manner. In NFAM1-expressing B cells, B cell antigen receptor stimulation induced NFAM1 translocation to lipid raft, and NFAM1 co-crosslinking augmented B cell antigen receptor signaling. The results suggest that NFAM1 modulates B cell signaling through its ITAM, which regulates B cell development.

Fig. 1.

Strategy of NACS and cloned genes. (A) Recipient cell line for NACS. The NFAT-GFP reporter cell line, 43–1, was stimulated by immobilized anti-TCR mAb (thick line) or left unstimulated (thin line), and it was analyzed for GFP expression by FACS. (B) Representative flow cytometric profiles during NACS screening. Clone 43–1 cells were transfected with a CD8-chimeric library, stimulated by crosslinking with anti-CD8, and analyzed for GFP expression before purification (Upper) and after three cycles of purification (Lower) by flow cytometry. (C) The number of cloned genes from the cDNA library of splenocytes and NK cells. Each number represents distinct clones. The number of analyzed clones was >10-fold of these numbers due to multiple identical clones.

Fig. 2.

Structure and expression of NFAM1. (A) Amino acid sequences of mouse and human NFAM1. Single and double underlines indicate signal peptide and transmembrane regions, respectively. Bold letters indicate ITAM. The cloned region of mouse NFAM1 by NACS is residues 207–3′UT. (B) Northern blot analysis of NFAM1 transcripts in various tissues. A total of 10 μg of total RNA was analyzed with an NFAM1 C-terminal probe (369 bp, residues 160–3′UT). (C) Quantitative real-time PCR analysis of NFAM1 mRNA in B cell subpopulations. B cell subpopulations in BM and spleen are defined as follows: pro-B, B220⁺ CD43⁺; pre-B, B220⁺ CD25⁺; splenic immature-B, B220⁺ IgM^high IgD^low; splenic mature-B, B220⁺ IgM^low IgD^high. Relative mRNA expression of NFAM1 in each population is shown as the ratio to the expression in splenic mature B cells. Data represent means of triplicate experiments.

Fig. 3.

Cell activation through NFAM1 and the requirement of ITAM for NFAT activation. (A) Clone 43–1 cells expressing CD8-NFAM1 (wild type, WT; Y177 mutant, Y177F; Y188 mutant, Y188F) were stimulated with immobilized anti-CD8 (5 μg/ml) or anti-TCR (1 μg/ml) for 12 h and analyzed for GFP expression. (B) Clone 43–1 transfectants expressing Flag-NFAM1 (WT, Y177F, and Y188F) were stained with anti-Flag (Left). These cells were stimulated with immobilized anti-Flag (10 μg/ml; thick line) for 12 h, and GFP expression was analyzed (Right). (C) IL-2 production in transfectants expressing CD8-NFAM1 (Upper) or Flag-NFAM1 (Lower). Transfectants were stimulated with control Ab or immobilized anti-CD8 or anti-Flag for 24 h. (D) Ca²⁺ mobilization in CD8-NFAM1-transfected B cells by crosslinking with anti-CD8. IIA1.6 B cells lacking FcγR expression were transfected with CD8-NFAM1 or CD8 vector and Ca²⁺ flux was measured upon stimulation with anti-CD8 or anti-Ig Ab. Arrows indicate time of Ab addition.

Fig. 4.

ITAM-dependent association of ZAP-70/Syk with NFAM1. (A) NFAM1 phosphorylation and ZAP-70 recruitment upon CD8-NFAM1 crosslinking. T cells expressing CD8-NFAM1 (WT, Y177F, and Y188F) were stimulated (+) with anti-CD8 plus anti-mouse Ig Ab for 2 min or were left unstimulated (–). Cell lysates were immunoprecipitated with anti-CD8 and were blotted with the indicated Abs. (B) Binding analysis of tyrosine-phosphorylated ITAM peptides of human (h) and mouse (m) NFAM1 and CD3ζ to ZAP 70 and Syk. Total cell lysate of splenocytes was mixed with each phosphorylated ITAM peptide of which the N terminus was biotinylated at indicated concentrations. The phosphorylated peptides were isolated with avidin-Sepharose and the protein–peptide complexes were electrophoresed and blotted with each Ab. Whole spleen cell lysate (SPL) was used as control.

Fig. 5.

Biochemical analysis of cell-surface NFAM1 and augmentation of BCR signaling by NFAM1. (A) Analysis of cell-surface NFAM1 on transfectants. T cell hybridoma expressing C-terminal Flag-NFAM1 was surface-biotinylated and cell lysates were immunoprecipitated (IP) with anti-Flag. Immunoprecipitates were treated (+) or not treated (–) with N-glycosidase F and analyzed under reducing conditions (Upper). The same membrane was reblotted with anti-Flag M2 mAb (Lower). Arrowheads indicate NFAM1. (B) Analysis of endogenous NFAM1 on the cell surface of splenocytes. Splenocytes were analyzed similar to A (Upper). The same membrane was reblotted with anti-NFAM1 Ab (Lower). (C) Co-crosslinking of NFAM1 and BCR enhances tyrosine phosphorylation. Flag-NFAM1- or mock-transfected WEHI-231 cells were stimulated with anti-IgM-biotin and anti-Flag-biotin followed by streptavidin. Total cell lysates were analyzed by Western blotting with anti-PY Ab. Arrowheads indicate particularly augmented phosphorylated proteins. (D) Translocation of NFAM1 to lipid raft upon BCR stimulation. Triton X-100 cell lysates of NFAM1-transfected B cells with (stim.) or without (unstim.) BCR stimulation. BCR stimulation was fractionated on sucrose density gradient centrifugation. Each fraction was analyzed by blotting with anti-NFAM1 or anti-Lyn.

Fig. 6.

Impaired B cell development in BM chimera and NFAM1-Tg mice. (A and B) Analysis of NFAM1-expressing BM chimera. BM cells from Ly5.1 mice were infected with retrovirus vector (mock), wild-type (WT), or Y177 and Y188 double-mutated NFAM1 (DM), and were transferred into lethally irradiated host mice. BM cells (A) and splenocytes (B) were stained with anti-Ly5.1 and indicated mAbs. All data gated for Ly5.1⁺ and GFP⁺ cells are shown. (C) Impairment of B cell development in NFAM1-Tg mice. BM, spleen, thymus, and peritoneum from NFAM1-Tg mice were analyzed by FACS. Percentages of cells in each population are shown. Data from only one Tg line (Tg.10) are shown and similar data were obtained with another line (Tg.7).